Tag: hypospadias

The resulting hypospadias phenotypes in male and female mice prenatally exposed to DES

2018 Study Abstract

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development (DSDs).

Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra.

This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol (DES), at a high, clinically relevant dose, and a low, previously untested dose, administered via water.

The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency.

Females exhibited hypospadias and urethral-vaginal fistula.

These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

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The current study describes the types of adult penile malformations induced developmentally by DES (hypospadias) in cohorts of mice treated with DES over the age range of E12 to P20, but examined in adulthood when enduring malformations are present

2016 Study Abstract

Hypospadias is a common malformation whose etiology is based upon perturbation of normal penile development. The mouse has been previously used as a model of hypospadias, despite an unacceptably wide range of definitions for this malformation.

The current paper presents objective criteria and a definition of mouse hypospadias. Accordingly, diethylstilbestrol (DES) induced penile malformations were examined at 60 days postnatal (P60) in mice treated with DES over the age range of 12 days embryonic to 20 days postnatal (E12 to P20). DES-induced hypospadias involves malformation of the urethral meatus, which is most severe in DES E12-P10, DES P0-P10 and DES P5-P15 groups and less so or absent in the other treatment groups. A frenulum-like ventral tether between the penis and the prepuce was seen in the most severely affected DES-treated mice. Internal penile morphology was also altered in the DES E12-P10, DES P0-P10 and DES P5-P15 groups (with little effect in the other DES treatment groups). Thus, adverse effects of DES are a function of the period of DES treatment and most severe in the P0 to P10 period. In “estrogen mutant mice” (NERKI, βERKO, αERKO and AROM+) hypospadias was only seen in AROM+ male mice having genetically-engineered elevation is serum estrogen. Significantly, mouse hypospadias was only seen distally at and near the urethral meatus where epithelial fusion events are known to take place and never in the penile midshaft, where urethral formation occurs via an entirely different morphogenetic process.

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Prenatal diethylstilbestrol induces malformation of the external genitalia of male and female mice and persistent second-generation developmental abnormalities of the external genitalia in two mouse strains

2014 Study Abstract

Potential trans-generational influence of diethylstilbestrol (DES) exposure emerged with reports of effects in grandchildren of DES-treated pregnant women and of reproductive tract tumors in offspring of mice exposed in utero to DES.

Accordingly, we examined the trans-generational influence of DES on development of external genitalia (ExG) and compared effects of in utero DES exposure in CD-1 and C57BL/6 mice injected with oil or DES every other day from gestational days 12 to 18. Mice were examined at birth, and on 5-120 days postnatal to evaluate ExG malformations. Of 23 adult (>60 days) prenatally DES-exposed males, features indicative of urethral meatal hypospadias (see text for definitions) ranged from 18% to 100% in prenatally DES-exposed CD-1 males and 31% to 100% in prenatally DES-exposed C57BL/6 males. Thus, the strains differed only slightly in the incidence of male urethral hypospadias. Ninety-one percent of DES-exposed CD-1 females and 100% of DES-exposed C57BL/6 females had urethral-vaginal fistula. All DES-exposed CD-1 and C57BL/6 females lacked an os clitoris. None of the prenatally oil-treated CD-1 and C57BL/6 male and female mice had ExG malformations. For the second-generation study, 10 adult CD-1 males and females, from oil- and DES-exposed groups, respectively, were paired with untreated CD-1 mice for 30 days, and their offspring evaluated for ExG malformations. None of the F1 DES-treated females were fertile. Nine of 10 prenatally DES-exposed CD-1 males sired offspring with untreated females, producing 55 male and 42 female pups. Of the F2 DES-lineage adult males, 20% had exposed urethral flaps, a criterion of urethral meatal hypospadias. Five of 42 (11.9%) F2 DES lineage females had urethral-vaginal fistula. In contrast, all F2 oil-lineage males and all oil-lineage females were normal.

Thus, prenatal DES exposure induces malformations of ExG in both sexes and strains of mice, and certain malformations are transmitted to the second-generation.

Abstract

BACKGROUNDDiethylstilbestrol (DES) is a synthetic estrogen that was widely prescribed to pregnant women before 1971. DES increases the risk of breast cancer in women who took the drug and the risk of reproductive tract abnormalities in their offspring. Dutch investigators have reported a 20-fold increase in risk of hypospadias among sons of women who were exposed to DES in utero. We assessed this relation in data from an ongoing study of DES-exposed persons.

METHODS
Several U.S. cohorts of women with documented exposure in utero to DES have been followed by mailed questionnaires since the 1970s. Comparison subjects are unexposed women of the same ages. In 1997, participants were asked about congenital abnormalities in their children. We calculated prevalence odds ratios for the risk of hypospadias in sons of exposed mothers relative to sons of unexposed mothers using generalized estimating equations to adjust for multiple sons per mother and controlling for maternal age at the son’s birth.

RESULTS
We obtained data from 3916 exposed and 1746 unexposed women. These women reported a total of 13 liveborn sons with hypospadias (10 exposed, 3 unexposed). The prevalence odds ratio for risk of hypospadias among the exposed was 1.7 (95% confidence interval = 0.4-6.8).

CONCLUSIONS
Our findings do not support a greatly increased risk of hypospadias among the sons of women exposed to DES in utero, as has been previously reported.

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These 2005 findings confirm an increased risk of hypospadias in the sons of women exposed in utero to DES and the transgenerational effect of DES

Abstract

Transgenerational effects of diethylstilbestrol (DES) have been reported in animals and humans. Alerted by two case reports, Klip H et al conducted a cohort study of all sons of a Dutch cohort of 16 284 women with a diagnosis of fertility problems and obtained a 67% response rate; their findings suggest an increased risk of hypospadias in the sons of women exposed in utero to DES. The mothers of 205 boys reported DES exposure in utero, and four of these boys were reported to have hypospadias. This defect was reported in only 8 of the remaining 8729 sons (prevalence ratio 21.3 (95% confidence interval (CI) 6.5–70.1)).

In a retrospective study, we analysed 32 406 computerized obstetrical and pediatric files at Port-Royal Maternity Center, covering births from 1 January 1993 to 31 December 2002. We compared the prevalence rate of hypospadias among 17 633 boys of mothers with and without in utero DES exposure.

The mothers of 240 boys had reported DES exposure in utero. Three (1.23%) were reported to have hypospadias. Obstetric records of the remaining 17 393 children reported hypospadias for only 44(0.5%◦) (prevalence ratio 4.99 (95% CI 1.2–16.8, p = 0.02)). All cases of hypospadias were medically confirmed by the pediatric files.

The lower prevalence ratio in our study (4.99) than in the Dutch cohort (21.3) is probably due to the difference in the underlying populations: the Dutch cohort was selected for the criteria of infertility and is thus necessarily different from our general population.

Klip’s data came from questionnaires addressed to women who had consulted for infertility and been previously enrolled in a cohort. The analysis was based on a 67% response rate. We note that the 95% CI of the relative risk (RR) in the Klip study (6.5–70.1) is compatible with the RR in the present study. On the basis of both the studies, the true RR may range between 6.5 (lower range of Dutch study) and 16.8 (upper range of our study) and, given the possible bias of the Dutch study, the true RR may well be lower than 16.8.

Our study examined a large continuous series of woman who gave birth at Port Royal. During the study period, women were systematically asked about their own in utero exposure to DES. All of the obstetrical and pediatric records of our population were analysed.

The prevalence of hypospadias in our control series was 2.5 per 1000, which corresponds to the prevalence in the general French population −2.89 per 1000 male newborns. The prevalence in the control series for Klip’s population was only 0.9 per 1000.

The importance of understanding the mechanism of hypospadias warrants additional studies. Van Tongeren et al. point out that, especially in urban areas, mothers’ occupations (such as hairdressing and working in beauty salons) can increase exposure to potential endocrinedisrupting chemicals Van Tongeren et al (2002). Our study does not enable us to determine whether the mothers of sons with hypospadias were exposed to higher levels of or at higher rates to such chemicals.

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Prenatal diethylstilbestrol induces malformation of the external genitalia of male and female mice and persistent second-generation developmental abnormalities of the external genitalia in two mouse strains

2014 Study Abstract

Potential trans-generational influence of diethylstilbestrol (DES) exposure emerged with reports of effects in grandchildren of DES-treated pregnant women and of reproductive tract tumors in offspring of mice exposed in utero to DES.

Accordingly, we examined the trans-generational influence of DES on development of external genitalia (ExG) and compared effects of in utero DES exposure in CD-1 and C57BL/6 mice injected with oil or DES every other day from gestational days 12 to 18. Mice were examined at birth, and on 5-120 days postnatal to evaluate ExG malformations. Of 23 adult (>60 days) prenatally DES-exposed males, features indicative of urethral meatal hypospadias (see text for definitions) ranged from 18% to 100% in prenatally DES-exposed CD-1 males and 31% to 100% in prenatally DES-exposed C57BL/6 males. Thus, the strains differed only slightly in the incidence of male urethral hypospadias. Ninety-one percent of DES-exposed CD-1 females and 100% of DES-exposed C57BL/6 females had urethral-vaginal fistula. All DES-exposed CD-1 and C57BL/6 females lacked an os clitoris. None of the prenatally oil-treated CD-1 and C57BL/6 male and female mice had ExG malformations. For the second-generation study, 10 adult CD-1 males and females, from oil- and DES-exposed groups, respectively, were paired with untreated CD-1 mice for 30 days, and their offspring evaluated for ExG malformations. None of the F1 DES-treated females were fertile. Nine of 10 prenatally DES-exposed CD-1 males sired offspring with untreated females, producing 55 male and 42 female pups. Of the F2 DES-lineage adult males, 20% had exposed urethral flaps, a criterion of urethral meatal hypospadias. Five of 42 (11.9%) F2 DES lineage females had urethral-vaginal fistula. In contrast, all F2 oil-lineage males and all oil-lineage females were normal.

Thus, prenatal DES exposure induces malformations of ExG in both sexes and strains of mice, and certain malformations are transmitted to the second-generation.

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Hypospadias in sons of women exposed to diethylstilbestrol: a true trans-generational effect?

2005 Study Abstract

In May 2005, Pons et al. reported on an increased risk of hypospadias in male children of women exposed to diethylstilbestrol (DES) in utero. The authors have retrospectively reviewed the electronic files from 17 633 deliveries of male neonates in a 10-year period. The mothers of 240 male neonates had reported in utero DES exposure, three of whom (1.23%) presented with hypospadias vs 44/17 393 (0.5%) in the remaining male neonates (from non-DES-exposed mothers). The authors conclude that there is an increased risk of hypospadias in the male children of women exposed in utero to DES due to the transgenerational effects of DES. Although these results apparently compare favourably with the initial Dutch cohort, we would like to address the authors with our concerns regarding the interpretation of these additional data.

In utero DES exposure has been associated not only with an increased risk of preterm labour but also with an increased risk of intra-uterine growth retardation (IUGR). In turn, an increased risk of cryptorchidism and hypospadias has been associated with decreased birth weight. One might expect a higher rate of IUGR in the subgroup of neonates of women exposed in utero to DES compared with the control group in the Parisian cohort, as previously observed in the Dutch one. This information, essential to the interpretation of the data, may avoid causing the patients undue concern about hypothetical transgenerational adverse effects of DES (i.e. genetic or epigenetic changes in either germ or somatic cells).

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2016 Study Abstract

Hypospadias, an abnormality affecting the penile urethra, is one of the most prevalent congenital malformations afflicting human males. The morphology of hypospadias is markedly different in humans versus mice reflecting substantial differences in penile development in humans and mice. Estrogens such as diethylstilbestrol (DES) elicit mouse penile malformations, but the types of penile abnormalities differ depending on whether DES treatment is prenatal or neonatal.

A thorough investigation of the effects of DES over a wide age range of treatment may

Penises of all 5 groups of DES-treated mice were reduced in size, which was confirmed by morphometric analysis of internal penile structures, and are presumably mediated via signaling through estrogen receptors alpha and/or beta (ERα and ERβ), which have been previously detected in all of the structures affected by DES.

The most profound effects were seen in the DES E12-P10, DES P0-P10, and DES P5-P15 groups, thus defining a DES “programming window”.

For all parameters, DES treatment from P10-P20 showed the most mild of effects.

Adverse effects of DES on the MUMP cartilage and erectile bodies observed shortly after the last DES injection reverted to normality in the DES P5-P15, but not in the E12-P10 and P0-P10 groups, in which MUMP cartilage and erectile body malformations persisted into adulthood, again emphasizing a “window of susceptibility” in the early neonatal period.

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Scanning electron micrographs of human fetal penises at 7 and 10 weeks of gestation. In (A) note the prominent urethral groove. In (B) the edges of the urethral groove are fusing in the midline to form the urethra, but the distal urethral groove is still widely open. Featured image credit PMC4803596/figure/F3.

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2002 Study Abstract

BACKGROUND
Transgenerational effects of diethylstilbestrol (DES) have been reported in animals, but effects in human beings are unknown. Alerted by two case reports, we aimed to establish the risk of hypospadias in the sons of women who were exposed to DES in utero.

METHODS
We did a cohort study of all sons of a Dutch cohort of 16284 women with a diagnosis of fertility problems. We used a mailed questionnaire assessing late effects of fertility treatment to identify boys with hypospadias. We compared the prevalence rate of hypospadias between boys with and without maternal DES exposure in utero.

FINDINGS
16284 mothers (response rate 67%) reported 8934 sons. The mothers of 205 boys reported DES exposure in utero. Four of these children were reported to have hypospadias. In the remaining 8729 children, only eight cases of hypospadias were reported (prevalence ratio 21.3 [95% CI 6.5-70.1]). All cases of hypospadias were medically confirmed. Maternal age or fertility treatment did not affect the risk of hypospadias. Children conceived after assisted reproductive techniques such as in-vitro fertilisation were not at increased risk of hypospadias compared with children conceived naturally (1.8, 0.6-5.7).

INTERPRETATION
Our findings suggest an increased risk of hypospadias in the sons of women exposed to DES in utero. Although the absolute risk of this anomaly is small, this transgenerational effect of DES warrants additional studies.

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Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study

2011 Study Abstract

Prenatal diethylstilbestrol (DES)-exposed mice have raised the suspicion of a transgenerational effect in the occurrence of genital malformation in males. This nationwide cohort study in collaboration with a French association of DES-exposed women studied 529 families and showed that a significant proportion of boys born to DES daughters exhibited hypospadias with no other molecular defects identified.

Although the role of fetal androgens is crucial to male genital development during the first trimester of pregnancy, defects in the synthesis or molecular action of testosterone are rare in isolated hypospadias (Hypospadias may be a multifactorial defect arising from genetic, hormonal, and environmental factors). It has been hypothesized that changes in androgen/estrogen balance due to endogenous or exogenous hormonal factors during the critical period of penile and urethral development contribute to this malformation.

Men who were exposed in utero to diethylstilbestrol (DES), a synthetic estrogen, may exemplify the effects of environmental chemicals with endocrine-disrupting activity on genital development (DES was prescribed for pregnant women from the late 1930s to the 1970s in the mistaken belief that it would prevent miscarriage or premature birth). Unfortunately, DES was found to be not only ineffective but also harmful. Daughters born from DES-related pregnancies often show abnormalities in the Müllerian structures and have elevated risks of peripubertal vaginal and cervical clear-cell adenocarcinoma, fertility problems, ectopic pregnancies, miscarriages, and premature births (The risk of reproductive tract abnormalities also appears to be increased for DES sons, who may present hypoplastic testis, epididymal cysts, cryptorchidism, or hypospadias).

After several studies in animals, a question emerged as to whether the harmful effects of DES can be “transmitted” to subsequent generations. Newbold and colleagues reported an increased incidence of reproductive tract tumors in male and female descendants of mice developmentally exposed to DES. In the human, Klip et al. reported an increased risk of hypospadias in sons of DES daughters in a cohort of women diagnosed with fertility problems. Other studies either confirmed or questioned these results. However, the direct implication of DES in the occurrence of hypospadias remains debatable, since many other uncontrolled factors, especially environmental and genetic, are implicated in this malformation. We studied the prevalence of hypospadias in the grandsons of DES-treated and -untreated women and ruled out other environmental and genetic factors that could have been associated with the malformation in these patients.

A nationwide cohort study was conducted in collaboration with a French association of DES-treated women (HHORAGES Association). The reason the women joined this association was not the presence of hypospadias in the second or third generation but rather psychological disturbances, vaginal and cervical clear-cell adenocarcinomas, miscarriages, and other abnormalities. Five hundred twenty-nine families were included. All of the second- and third-generation offspring were accounted for and included in the study. No one declined to participate. DES exposure was reported in 1,000 out of 1,180 pregnancies. The featured image details the patient groups. The clinical diagnosis of hypospadias was standardized and based on a detailed operative report or direct clinical examination by a pediatrician and/or urologist. The malformation was characterized as severe (proximal, penoscrotal) or nonsevere (glandular, subcoronal, distal, midshaft). Each mother with a hypospadiac son was contacted and responded to a short questionnaire validated in Europe for data collection (no. QLK4-1999-01422) to determine whether other occupational exposure had occurred during the pregnancy. To exclude a defect of the androgen pathway, we performed molecular analysis of the genes known to be associated with hypospadias such as androgen receptor (AR), 5α reductase (srd5A2), and MAMLD1 genes in DNA from peripheral blood. The local university hospital ethics committee approved this study (ID RCB No. 2008-A00781-54), and each patient gave informed consent through the Hhorage Association.

The prevalence of hypospadias was low in boys unexposed to DES in utero (0/180), whereas it was high in the in utero–exposed boys (3.57%, 16/448, P=.02). In the third generation, the prevalence of hypospadias in boys born to DES daughters was high when compared with boys born to unexposed parents (8.2%, n = 8/97 vs. n = 0/360; P<.001). The hypospadiac patients of the second and third generations were not related. The results are summarized in the featured image.

Neither mutations nor polymorphisms of the AR and MAMLD1 genes were found among hypospadiac boys of the third generation. Only one polymorphism of the srd5A2 gene was detected (A49T) in a boy. The mothers of the third-generation affected boys indicated little environmental or occupational exposure to endocrine-disrupting chemicals during pregnancy (no professional activity, n = 2; sales clerk in a food or clothing shop, n = 3; office worker, n = 3), and such exposure was therefore unlikely to have contributed to the occurrence of hypospadias. Two mothers of hypospadiac sons exhibited hypoplastic or bifid uterus.

The main effect of DES is profound disturbance in the androgenic/estrogenic balance of animal and human fetuses since it has both estrogenic and antiandrogenic actions by competing with natural androgens for the ligand-binding domain of the androgen receptor. In utero exposure to DES during the critical period of reproductive tract development is known to induce genital malformation in mice. In utero–exposed sons show greater risks of structural urogenital abnormalities like hypospadias, epididymal cysts, micropenis, and cryptorchidism. The present study reinforces these data with a prevalence of hypospadias greater than 3%, although it should be noted that the second-generation population included only 180 controls from the same families since this series was specifically designed to study the third-generation boys.

More interesting is the hypothesis of a transgenerational effect of DES. Animal studies first suggested that DES might increase transgenerational susceptibility to malignant tumors of the female reproductive tract, presumably by damage to germ cells and abnormal imprinting. In human beings, DES exposure may also lead to permanently altered germ cells. The suggestion of a transgenerational effect of DES in human beings was based on the observation of a high prevalence of hypospadias, particularly with severe phenotypes, in the sons of women exposed to DES in utero.

But variations in the definition of the control population may explain the wide range of odds ratios reported in the literature to date. Palmer et al. reported a prevalence 6 times higher than that of Klip et al. The present study, which shows a high prevalence of hypospadias of various severities in the third generation, tried to limit this bias and included DES-free pregnancies and DES-exposed pregnancies from the same families. Nevertheless, two limitations should be noted:

DES-exposed women without problems were not included;

and the fertility status of the exposed and non-exposed couples, the age at pregnancy, and the parity for each women were variable, and this may have hidden fertility problems or greater use of contraception.

The low fertility rates of the DES sons may also be explained by other findings, such as severe psychotic disorders or oligospermia in cases of hypospadias with additional defects.

We did not identify any genetic or environmental factors that would have explained the hypospadias in DES grandsons. Our results thus raise the question of the mechanism through which DES causes adverse effects in subsequent generations. The frequency of transmission both observed in our series of hypospadiac grandsons and previously reported in generations examined for various disease states secondary to DES exposure is particularly high. This frequency of a transgenerational phenotype is such that a mutational event involving the DNA sequence could not be implicated. DES-induced changes in epigenetic background and alteration of DNA methylation could be significant factors in the susceptibility to disease development. The primordial germ cells undergo demethylation during migration and early colonization of the embryonic gonad, followed by remethylation starting at the time of sex determination in a sex-specific manner. The pregnant mother’s exposure to DES at the time of fetal sex determination appears to be sufficient to alter the remethylation of the germ line in the male fetus and permanently reprogram the imprinted pattern of DNA methylation. The transmission of multigenerational DES effects would thus occur through the paternal lineage. But our findings indicated that most of the third-generation hypospadiac boys were born to DES daughters. This agreed with previous studies (although paternal transmission of DES effects is not excluded). Epigenetic changes in the AR gene, transmitted through the DES daughter, could explain such a finding since the antiandrogen effect of DES is known to modify the phosphorylation level of AR.

The association between hypospadias in grandsons and uterine abnormalities in their mothers suggests other hypotheses for the transgenerational mechanisms of DES. First, DES daughters may have displayed disturbed hormonal balance during their reproductive life or placental malfunction that might have interfered with the genital development of a male fetus. Second, the estrogen receptor gene ERα and estrogen-responsive genes that contribute to the development of both female internal genitalia and hypospadias may also be involved since ERα is implicated in the induction of abnormalities after DES exposure. Last, the genes involved in the structural differentiation of both the female and male reproductive tracts may be altered by DES exposure. DES has been reported to delay expression of HOXa family genes during Müllerian duct development. DES could also interfere with HOX gene expression during penile formation.

For many authors, DES is an experimental environmental xenoestrogen. Despite the bias that could not be fully eliminated and the difficulty of extrapolating the risks of exposure (no monotonic dose-response relationship, varying effects depending on the timing of exposure in the developing organism, manifestations delayed until later in life), this clinical study strengthens the suspicion of the transgenerational effects of environmental endocrine disruptors.

Sources

Full text (free access) : Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study, CHU Montpellier and Universite Montpellier, France, hhorages, February 23, 2011.

Featured image : Detailed patient groups included in the study. In the second generation, the phenotype of affected boys was isolated hypospadias in all cases, severe in 12 cases (posterior or penoscrotal), and not severe in four cases (mild or anterior). In the third generation, the hypospadias was severe in five cases and not severe in three cases. Bilateral cryptorchidism was present in one case. *The size of the population was under 1,000, and the prevalence of hypospadias of about 1/1,000, as seen in the general population, could not be represented.